Method for reducing pest damage to corn by treating transgenic corn seeds with thiamethoxam pesticide

ABSTRACT

A method to protect corn against feeding damage by one or more pests includes the treatment of corn seed having a transgenic event that is targeted against at least one of the pests with thiamethoxam in an amount that is effective against the same or another of the one or more pests. Seeds having such protection are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional of U.S. patent application Ser.No. 60/238,406, filed on Oct. 6, 2000, and claims priority thereto.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to the control of pests thatcause damage to corn plants by their feeding activities, and moreparticularly to the control of such corn plant pests by the combinationof a corn seed having a transgenic event and the treatment of such seedwith a thiamethoxam pesticide prior to planting the seed.

(2) Description of the Related Art

Insects and related arthropods annually destroy an estimated 15% ofagricultural crops in the United States and even more than that indeveloping countries. In addition, competing weeds and parasitic andsaprophytic plants account for even more potential yield losses.

Some of this damage occurs in the soil when plant pathogens, insects andother such soil borne pests attack the seed after planting. In theproduction of corn, much of the rest of the damage is caused byrootworms—insect pests that feed upon or otherwise damage the plantroots; and by cutworms, European corn borers, and other pests that feedupon or damage the above ground parts of the plant. General descriptionsof the type and mechanisms of attack of pests on agricultural crops areprovided by, for example, Metcalf, in Destructive and Useful Insects,(1962); and Agrios, in Plant Pathology, 3rd Ed., Academic Press (1988).

Corn is the most important grain crop in the Midwestern United States.Among the most serious insect pests of corn in this region are thelarval forms of three species of Diabrotica beetles. These include theWestern corn rootworm, Diabrotica vergifera vergifera LeConte, theNorthern corn rootworm, Diabrotica berberi Smith and Diabrotica berberiLawrence, and the Southern corn rootworm, Diabrotica undecimpunctatahowardi Barber. In fact, more insecticide is used for the control ofcorn rootworm than for any other pest of corn, and the total acreagetreated is greater than for any other pest in the United States.

Corn rootworms (CRW) overwinter in the egg stage in fields where cornwas grown the previous season. The eggs hatch from late May throughJune. If a corn crop is not followed by another corn crop in thesubsequent year, the larvae will die. Accordingly, the impact of cornrootworm is felt most directly in areas where corn is systematicallyfollowed by corn, as is typical in many areas of the Midwestern UnitedStates.

After hatching, the larvae pass through three larval stages or instars,during which they feed on the corn root system. About three weeks isrequired for completion of the larval stage. Damage to the corn rootsystem caused by the feeding of larvae is the major cause of harvestlosses in corn due to corn rootworm. Corn plants that fall over andlodge in the field after weakening or destruction of a major part of theroot system are the cause of a major portion of this loss, since thislodged corn cannot be harvested by conventional mechanized machinery andis left in the field.

Following completion of larval development, the larvae transform intoimmobile pupae, and thence into the adult beetles that emerge from thesoil throughout the summer, with the period of emergence depending uponthe growing location. After emergence, the adult beetles feed for abouttwo weeks before the females start laying eggs. Initially, the adultsfeed predominantly in the same field from which they emerged, but laterwill migrate to other fields. Peak adult activity normally occurs in theU.S. Corn Belt during late July or early August in fields planted tocontinuous corn, but activity may peak later in first year or latematuring cornfields. Rootworm beetles begin depositing eggs incornfields approximately two weeks after they emerge. (For moreinformation, see, e.g., Corn Rootworms, Field Crops Pest ManagementCircular #16, Ohio Pest Management & Survey Program, The Ohio StateUniversity, Extension Division, Columbus, Ohio; available online atwww.ag.ohio-state.edu/˜ohioline/icm-fact/fc-16.html, Sep. 13, 2000; andMcGahen et al., Corn Insect Control: Corn Rootworm, PENpages number08801502, Factsheet available from Pennsylvania State University, StateCollege, PA, 1989).

In present conventional agricultural practice, in cases where cornfollows corn, it is normal for an insecticide to be applied to protectthe corn root system from severe feeding by rootworm larvae.Conventional practice is to treat for the adult beetles or to treat forthe larvae. Examples of conventional treatment formulations for adultbeetles include the application of carbaryl insecticides (e.g., SEVIN®80S at 1.0-2.0 lbs active/acre); fenvalerate or esfenvalerate (e.g.,PYDRIN® 2.4EC at 0.1 to 0.2 lbs active/acre, or ASANA® 0.66EC at 0.03 to0.05 lbs active/acre); malathion (57% E at 0.9 lbs active/acre);permethrin (e.g., AMBUSH® 2.0EC at 0.1 to 0.2 lbs active/acre, orPOUNCE® 3.2EC at 0.1 to 0.2 lbs active ingredient/acre); or PENNCAP-M®at 0.25-0.5 lbs active/acre.

To treat for CRW larvae, conventional practice is to apply a soilinsecticide either at or after planting, but preferably as close to egghatching as possible. Conventional treatments include carbofuraninsecticides (e.g., FURADAN® 15G at 8 oz/1000 ft of row); chloropyrifos(e.g., LORSBAN® 15G at 8 oz/1000 ft of row); fonophos (e.g., DYFONATE®20G at 4.5 to 6.0 oz/1000 ft of row); phorate (e.g., THIMET® 20G at 6oz/1000 ft of row); terbufos (e.g., COUNTER® 15G at 8 oz/1000 ft ofrow); or tefluthrin (e.g., FORCE® 3G at 4 to 5 oz/1 000 ft of row).

Many of the chemical pesticides listed above are known to be harmful tohumans and to animals in general. The environmental harm that thesepesticides cause is often exacerbated due to the practice of applyingthe pesticides by foliar spraying or direct application to the surfaceof the soil. Wind-drift, leaching, and runoff can cause the migration ofa large fraction of the pesticide out of the desired zone of activityand into surface waters and direct contact with birds, animals andhumans.

Because of concern about the impact of chemical pesticides on publichealth and the health of the environment, significant efforts have beenmade to find ways to reduce the amount of chemical pesticides that areused. Recently, much of this effort has focused on the development oftransgenic crops that are engineered to express insect toxicants derivedfrom microorganisms. For example, U.S. Pat. No. 5,877,012 to Estruch etal. discloses the cloning and expression of proteins from such organismsas Bacillus, Pseudomonas, Clavibacter and Rhizobium into plants toobtain transgenic plants with resistance to such pests as blackcutworms, armyworms, several borers and other insect pests. PublicationWO/EP97/07089 by Privalle et al. teaches the transformation ofmonocotyledons, such as corn, with a recombinant DNA sequence encodingperoxidase for the protection of the plant from feeding by corn borers,earworms and cutworms. Jansens et al., in Crop Sci., 37(5):1616-1624(1997), reported the production of transgenic corn containing a geneencoding a crystalline protein from Bacillus thuringiensis (Bt) thatcontrolled both generations of the European corn borer. U. S. Pat. Nos.5,625,136 and 5,859,336 to Koziel et al. reported that thetransformation of corn with a gene from B. thuringiensis that encodedfor delta-endotoxins provided the transgenic corn with improvedresistance to European corn borer. A comprehensive report of fieldtrials of transgenic corn that expresses an insecticidal protein from B.thuringiensis has been provided by Armstrong et al., in Crop Science,35(2):550-557 (1995).

It was known that wild-type Bt δ-endotoxins had low activity againstcoleopteran insects, and Kreig et al., in 1983, reported the firstisolation of a coleopteran-toxic B. thuringiensis strain. (See U.S. Pat.No. 4,766,203). U.S. Pat. Nos. 4,797,279 and 4,910,016, also disclosedwild-type and hybrid B. thuringiensis strains that produced proteinshaving some coleopteran activity. More recently, however, more precisegenetic engineering methods have shown promise in developing modified B.thuringiensis proteins that have significantly higher levels of cornrootworm activity than those produced by wild-type parents. (See, e.g.,WO 99/31248 to Ecogen, Inc. and Monsanto Company).

However, it is not known at present whether any transgenic event alonewill be sufficiently effective to protect corn from damage by cornrootworm in heavily infested fields that are dedicated to serial corn.In fact, the total control of corn rootworm damage by any one transgenicevent may not be desirable in the long term, because of the potentialfor the development of resistant strains of the target pest.

Another alternative to the conventional forms of pesticide applicationis the treatment of plant seeds with pesticides. The use of fungicidesto protect seeds from attack after planting, and the use of low levelsof insecticides for the protection of, for example, corn seed fromwireworm, has been used for some time. Seed treatment with pesticideshas the advantages providing for the protection of the seeds, whileminimizing the amount of pesticide that was required and limiting theamount of contact with the pesticide and the number of different fieldapplications that were necessary.

Other examples of the control of pests by applying insecticides directlyto plant seed are provided in, for example, U.S. Pat. No. 5,696,144,which discloses that the European corn borer caused less feeding damageto corn plants grown from seed treated with a 1-arylpyrazole compound ata rate of 500 g per quintal of seed than control plants grown fromuntreated seed. In addition, U.S. Pat. No. 5,876,739 to Turnblad et al.(and its parent, U. S. Pat. No. 5,849,320) disclose a method forcontrolling soil-borne insects which involves treating seeds with acoating containing one or more polymeric binders and an insecticide.This reference provides a list of insecticides that it identifies ascandidates for use in this coating and also names a number of potentialtarget insects. However, while the U.S. Pat. No. 5,876,739 patent statesthat treating corn seed with a coating containing a particularinsecticide protects corn roots from damage by the corn rootworm, itdoes not indicate or otherwise suggest that such treatment could be usedwith seed having a transgenic event.

The treatment of seed having a transgenic event with nitroimino- ornitroguanidino-compound pesticides has been mentioned (See, e.g., WO99/35913), however, no guidance has been found as to the potentialutility or efficacy of such treatments, or the details of how suchtreatments might be effected—such as the amounts of active ingredientthat would be necessary per unit amount of seed—and no examples thatwould give reason to believe that the proposed treatments would actuallyprovide suitable protection.

Therefore, although recent developments in genetic engineering of plantshave improved the ability to protect plants from pests without usingchemical pesticides, and while such techniques as the treatment of seedswith pesticides have reducing the harmful effects of pesticides on theenvironment, numerous problems remain that limit the successfulapplication of these methods under actual field conditions. Accordingly,it would be useful to provide an improved method for the protection ofplants, especially corn plants, from feeding damage by pests. It wouldbe particularly useful if such method would reduce the requiredapplication rate of conventional chemical pesticides, and also if itwould limit the number of separate field operations that were requiredfor crop planting and cultivation.

BRIEF SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to a novel methodfor protecting a transgenic corn plant against feeding damage by one ormore pests, the method comprising providing a seed for the transgeniccorn plant which seed comprises a transgenic event having activityagainst at least one of the one or more pests; and treating the seedwith an effective amount of thiamethoxam pesticide.

The present invention is also directed to a novel seed of a transgeniccorn plant that provides increased resistance to the resulting cornplant against feeding damage by one or more pests, comprising atransgenic event having activity against at least one of the one or morepests, which seed has been treated with an effective amount ofthiamethoxam pesticide.

The present invention is also directed to a novel transgenic corn seedthat has been treated by the method of the present invention.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of an improved methodfor the protection of plants, especially corn plants, from feedingdamage by pests; the provision of such a method that reduces therequired application rate of conventional chemical pesticides; and alsothe provision of such a method that limits the number of separate fieldoperations that were required for crop planting and cultivation.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been discovered thatcorn plants can be protected against feeding damage by one or more pestsby a method that includes providing a corn seed having a transgenicevent that has activity against at least one of the pests and thentreating the transgenic corn seed with an effective amount ofthiamethoxam pesticide. For example, in preferred embodiments it hasbeen found that the combination of a transgenic event having activityagainst corn rootworm and treatment of the seed with thiamethoxamprovides unexpectedly synergistic advantages to seeds having suchtreatment, including unexpectedly superior efficacy for protectionagainst damage to the resulting corn plant by corn rootworm. Inparticular, it is believed that corn seeds having a transgenic eventthat causes the production of CryBb.11231 endotoxin in combination withthe treatment of such seeds with thiamethoxam would be unexpectedlysuperior to either the transgenic event alone, or to treatment withithiamethoxam alone, in protecting resulting corn plants against moresevere levels of damage by corn rootworm—levels of damage that are knownto reduce corn yield.

Corn plants and seeds that have been engineered to include exogenousgenes derived from Bacillus thuringiensis that encode for the expressionof Cry3 δ-endotoxins having activity against Coleopteran pests areknown, as are methods for the treatment of seeds (even some transgenicseeds) with pesticides. However, it had not been realized until thepresent invention that certain effective amounts of thiamethoxam couldbe used to treat corn seeds having such Cry3 events, with the resultthat the combination would be unexpectedly superior in increasing theefficacy of both the pesticide and the transgenic event, and wouldprovide the additional advantages of increasing the ability to matchpesticidal activity against pest pressure, decreasing cost of treatmentand/or application, increasing safety of seed handling, and decreasingenvironmental impact of either or both the event and the pesticide.

In particular, it is believed that in preferred embodiments thetreatment of transgenic corn seeds that are capable of expressingcertain modified Cry3Bb proteins with from about 100 gm to about 400 gmof thiamethoxam per 100 kg of seed can provide unexpectedly superiorprotection against corn rootworm. In addition, it is believed that suchcombinations are also effective to protect the emergent corn plantsagainst damage by black cutworm. The seeds of the present invention arealso believed to have the property of decreasing the cost of pesticideuse, because less of the pesticide can be used to obtain a requiredamount of protection than if the innovative method is not used.Moreover, because less pesticide is used and because it is applied priorto planting and without a separate field application, it is believedthat the subject method is therefore safer to the operator and to theenvironment, and is potentially less expensive than conventionalmethods.

When it is said that some effects are “synergistic”, it is meant toinclude the synergistic effects of the combination on the pesticidalactivity (or efficacy) of the combination of the transgenic event andthe pesticide. However, it is not intended that such synergistic effectsbe limited to the pesticidal activity, but that they should also includesuch unexpected advantages as increased scope of activity, advantageousactivity profile as related to type and amount of damage reduction,decreased cost of pesticide and application, decreased pesticidedistribution in the environment, decreased pesticide exposure ofpersonnel who produce, handle and plant corn seeds, and other advantagesknown to those skilled in the art.

The present invention also provides an advantage of increasing theability to match pesticidal activity against pest pressure. This refersto the ability to design the combination of the transgenic event and thepesticide treatment so that the seed or the resulting plant is providedwith effective pesticidal activity during the period when feedingpressure from the target pest on the seed or plant reaches its maximum.By way of example, when a pesticide such as thiamethoxam is applied to acorn seed having a corn rootworm transgenic event, the pesticide can beapplied in a coating designed to provide controlled release of thepesticide. The release rate can be selected so that the pesticideprovides protection against such other pests as are controlled bythiamethoxam at the post emergence stage of corn, while the transgenicevent provides corn rootworm protection at a later stage of plantdevelopment—when such protection is needed.

As used herein, the terms “pesticidal effect” and “pesticidal activity”,or “activity” refer to a toxic effect against a pest. The terms“activity against (one or more) pests”, also have the same meaning. Whenit is said that a seed or plant is “protected against feeding damage byone or more pests”, it is meant that such seed or plant possesses afeature having direct or indirect action on one or more pests thatresults in reduced feeding damage by such pest or pests on the seeds,roots, shoots and foliage of plants having such feature as compared tothe feeding damage caused under the same conditions to plants not havingsuch feature. Such direct or indirect actions include inducing death ofthe pest, repelling the pest from the plant seeds, roots, shoots and/orfoliage, inhibiting feeding of the pest on, or the laying of its eggson, the plant seeds, roots, shoots and/or foliage, and inhibiting orpreventing reproduction of the pest.

The term “insecticidal activity” has the same meaning as pesticidalactivity, except it is limited to those instances where the pest is aninsect. Except where specifically noted, when the term “pesticide” isused herein, that term refers to a chemical pesticide that is suppliedexternally to the seed, and it is not meant to include active agentsthat are produced by the particular seed or the plant that grows fromthe particular seed. However, the terms “pesticidal activity” and“insecticidal activity” can be used with reference to the activity ofeither, or both, an externally supplied pesticide and/or an agent thatis produced by the seed or the plant.

One feature of the present invention is a seed of a transgenic cornplant. As used herein, the terms “transgenic corn plant” mean a cornplant or progeny thereof derived from a transformed corn plant cell orprotoplast, wherein the plant DNA contains an introduced exogenous DNAmolecule not originally present in a native, non-transgenic plant of thesame strain.

The transgenic corn seed is one that contains an exogenous gene thatencodes a pesticidal protein. Pesticidal proteins of this type aredescribed by Schnepf et al., in Microbiology & Molecular BiologyReviews, 62:775-806 (1998), and by ffrench-Constant and Bowen, in CMSLCell. Mol. Life Sci., 57:828-833 (2000). In one application of theinvention, the pesticidal protein is an insecticidal protein.

It is preferred that the seed contains an exogenous gene derived from astrain of Bacillus thuringiensis, and in particular, it is preferredthat the exogenous gene is one that encodes an insecticidal δ-endotoxinderived from B. thuringiensis. Such δ-endotoxins are described in WO99/31248 and U.S. Pat. No. 6,063,597, and include the Cry3 toxins.Nucleic acid segments that encode modified B. thuringiensiscoleopteran-toxic crystal proteins that are useful in the presentinvention are described in U.S. Pat. No. 6,060,594, and insect resistanttransgenic plants that include nucleic acid sequences that encode suchinsecticidal proteins are discussed in U.S. Pat. No. 6,023,013. It ispreferred that the δ-endotoxins of the present invention include theCry3B proteins, and even more preferred that the δ-endotoxins includethe coleopteran-active Cry3Bb proteins. The nomenclature of the B.thuringiensis insecticidal crystal proteins was set forth by Höfte andWhitely, Microbiol. Rev., 53:242-255, 1989. This nomenclature has beenrevised, and the revised nomenclature can be found athttp://epunix.biols.susx.ac.uk/Home/Neil-Crickmore/Bt/index.html. Therevised nomenclature will be used herein to describe transgenic eventfeatures and the δ-endotoxin proteins encoded by the transgenic event.

When the terms “transgenic event” are used herein, such terms are meantto refer to the genetically engineered DNA that is described above, butalso to include the protein(s) that are encoded by the modified gene. Atransgenic event in a corn seed, or corn plant, therefore, includes theability to express a protein. When it is said that a “transgenic eventhas activity against a pest”, it is to be understood that it is theprotein that is encoded by the gene that actually has such activity whenthe protein is expressed and brought into contact with the pest.

WO 99/31248 and U.S. Pat. Nos. 6,063,597 describe methods forgenetically engineering B. thuringiensis δ-endotoxin genes so thatmodified δ-endotoxins can be expressed. The modified δ-endotoxins differfrom the wild-type proteins by having specific amino acid substitutions,additions or deletions as compared with the proteins produced by thewild-type organism. Such modified δ-endotoxins are identified herein bythe use of an asterisk (*), or by reference to a specific protein by itsidentifying number. Thus, a genetically modified Cry3 δ-endotoxin wouldbe expressed as Cry3*, examples of which include, without limitation:Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234,Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239,Cry3Bb.11241, Cry3Bb.11242, and Cry3Bb.11098.

Some of the modified δ-endotoxins that were described in WO 99/31248 andin U.S. Pat. No. 6,063,597 were found to have enhanced activity againstcoleopteran insects, and in particular against Diabrotica spp.,including corn rootworm. As used herein, the terms “enhanced activity”refer to the increased insecticidal activity of a modified toxin ascompared with the activity of the same toxin without the amino acidmodifications when both are tested under the same conditions. Inparticular, it was found that Cry3* δ-endotoxins had enhanced activityagainst corn rootworm, and are therefore preferred for use in thepresent invention. More preferred are Cry3B* δ-endotoxins, and even morepreferred are Cry3Bb* δ-endotoxins. Even more preferred transgenicevents are those that comprise the ability to express the modifiedδ-endotoxins that are listed in the following table. Also shown in thetable are strains of transgenic B. thuringiensis that include genes forexpression of the respective novel endotoxins, and the date andaccession number of their deposit with the Agricultural Research ServiceCollection (NRRL) at 1815 N. University Street, Peoria, Ill. 91904.

ACCESSION NUMBER (NRRL STRAIN DEPOSIT DATE PROTEIN NUMBER) EG11230 May27, 1997 Cry3Bb.11230 B-21768 EG11231 May 27, 1997 Cry3Bb.11231 B-21769EG11232 May 27, 1997 Cry3Bb.11232 B-21770 EG11233 May 27, 1997Cry3Bb.11233 B-21771 EG11234 May 27, 1997 Cry3Bb.11234 B-21772 EG11235May 27, 1997 Cry3Bb.11235 B-21773 EG11236 May 27, 1997 Cry3Bb.11236B-21774 EG11237 May 27, 1997 Cry3Bb.11237 B-21775 EG11238 May 27, 1997Cry3Bb.11238 B-21776 EG11239 May 27, 1997 Cry3Bb.11239 B-21777 EG11241May 27, 1997 Cry3Bb.11241 B-21778 EG11242 May 27, 1997 Cry3Bb.11242B-21779 EH11098 Nov. 28, 1997 Cry3Bb.11098 B-21903

It has also been found that a preferred use of the present invention isfor reducing pest feeding damage when used in combination with seedshaving transgenic events that have certain levels of effectivenessagainst such pest. To illustrate which levels of effectiveness arepreferred, the following example will use the Iowa Root Rating Method(Hills and Peters, J. Econ. Entomol, 64:764-765, 1971), which measurescorn rootworm feeding damage to corn roots on a 1-6 scale. In therating, 1=no damage or only a few minor feeding scars; 2=feeding scarsevident but no roots eaten off to within 1½ inch of the plant; 3=severalroots eaten off to within 1½ inch of the plant, but never the equivalentof an entire node of roots is destroyed; 4=one root node completelydestroyed; 5=two root nodes completely destroyed; and 6=three or moreroot nodes destroyed. A destroyed root is defined as a root that hasbeen pruned to within 1½ inch of the base. Pruned roots do not have tooriginate from a single node, but all pruned roots must equal theequivalent of a full node to count as a destroyed node.

As used herein, a transgenic event is within the preferred range ofeffectiveness level against a target pest if that event reduces feedingdamage by that pest by a certain amount as compared with the same cropwithout the transgenic event, but does not prevent substantially alldamage by the target pest. For example, if 10% of transgenic cornsuffered corn rootworm damage of 4 or higher on the Iowa 1-6 Scale,while 80% of non-transgenic corn suffered damage of 4 or higher, then itcould be said that the damage to the transgenic corn was (10/80)×100=12.5% of that of the non-transgenic corn. For the purposes ofthe present invention, it will be understood that a transgenic event incorn is within the preferred range of effectiveness level if corn havingsuch event suffers from about 5% to about 50% of the damage suffered bynon-transgenic corn due to the same pest under the same conditions. Itis more preferred that corn having such transgenic event suffers fromabout 10% to about 40% of the damage suffered by non-transgenic corn bythe same pest under the same conditions, even more preferred is damageof from about 15% to about 30%, and yet more preferred is damage of fromabout 20% to about 30% of the damage suffered by non-transgenic corn bythe same pest under the same conditions. As used herein, when the term“about” is used to describe the degree of damage to corn, it is to beunderstood that the degree of damage can be above or below the limitsdescribed by as much as 1% or 2% and still be considered to be withinthe ranges described. By way of example, a level of 4.5% damage would beregarded as being “about 5%”.

Without wishing to be bound to this or any other theory, it is believedthat the pesticidal seed treatment can provide significant advantageswhen combined with a transgenic event that provides protection that iswithin the preferred effectiveness range against a target pest. Inaddition, it is believed that there are situations that are well knownto those having skill in the art, where it is advantageous to have suchtransgenic events within the preferred range of effectiveness.

The present invention also includes seeds and plants having more thatone transgenic event. Such combinations are referred to as “stacked”transgenic events. These stacked transgenic events can be events thatare directed at the same target pest, or they can be directed atdifferent target pests. In one preferred method, a seed having theability to express a Cry 3 protein also has the ability to express atleast one other insecticidal protein that is different from a Cry 3protein.

In another preferred method, the seed having the ability to express aCry 3 protein also has a transgenic event that provides herbicidetolerance. It is more preferred that the transgenic event that providesherbicide tolerance is an event that provides resistance to glyphosate,N-(phosphonomethyl) glycine, including the isopropylamine salt form ofsuch herbicide, even more preferred is the transgenic event that iseffective to provide the herbicide resistance of ROUNDUP READY® plantsand seeds available from Monsanto Co., St. Louis, Mo.

In the present method, a corn seed having a transgenic event is treatedwith thiamethoxam(3-[(2-chloro-5-thiazolyl)mehthyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiazin-4-imine;CAS RN 153719-23-4; and described in EP 580553, as well as in ThePesticide Manual, Id., at 896).

When an insecticide is described herein, it is to be understood that thedescription is intended to include salt forms of the insecticide as wellas any isomeric and/or tautomeric form of the insecticide that exhibitsthe same insecticidal activity as the form of the insecticide that isdescribed.

The insecticides that are useful in the present method can be of anygrade or purity that pass in the trade as such insecticide. Othermaterials that accompany the insecticides in commercial preparations asimpurities can be tolerated in the subject methods and compositions, aslong as such other materials do not destabilize the composition orsignificantly reduce or destroy the activity of any of the insecticidecomponents or the transgenic event against the target pest(s). One ofordinary skill in the art of the production of insecticides can readilyidentify those impurities that can be tolerated and those that cannot.

It has been found that the present method is useful to protect seeds andplants against a wide array of agricultural pests, including insects,mites, fungi, yeasts, molds, bacteria, nematodes, weeds, and parasiticand saprophytic plants.

When an insect is the target pest for the present invention, such pestsinclude but are not limited to:

from the order Lepidoptera, for example,

Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabamaargillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp,Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella,Carposina nipponensis, Chilo spp., Choristoneura spp., Clysiaambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp.,Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydiaspp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp.,Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp.,Ostrinia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea,Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp.,Tortrix spp., Trichoplusia ni and Yponomeuta spp.;

from the order Coleoptera, for example,

Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp.;

-   from the order Orthoptera, for example,

Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae,Locusta spp., Periplaneta ssp., and Schistocerca spp.;

-   from the order Isoptera, for example,

Reticulitemes ssp;

-   from the order Psocoptera, for example,

Liposcelis spp.;

-   from the order Anoplura, for example,

Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. andPhylloxera spp.;

-   from the order Mallophaga, for example,

Damalinea spp. and Trichodectes spp.;

-   from the order Thysanoptera, for example,

Franklinella spp., Hercinothrips spp., Taeniothrips spp., Thrips palmi,Thrips tabaci and Scirtothrips aurantii;

-   from the order Heteroptera, for example,

Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp.,Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodniusspp., Sahlbergella singularis, Scotinophara spp. and Triatoma spp.;

-   from the order Homoptera, for example,

Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lacanium comi, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nehoteftix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri;

-   from the order Hymenoptera, for example,

Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpiniapolytoma, Hoplocampa spp., Lasius sppp., Monomorium pharaonis,Neodiprion spp, Solenopsis spp. and Vespa ssp.;

-   from the order Diptera, for example,

Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphoraerythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebraspp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilusspp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp.,Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseoliaspp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletispomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. andTipula spp.,

-   from the order Siphonaptera, for example,

Ceratophyllus spp. und Xenopsylla cheopis and from the order Thysanura,for example,

Lepisma saccharina.

It has been found that the present invention is particularly effectivewhen the insect pest is a Diabrotica spp., and especially when the pestis Diabrotica virgifera, Diabrotica barberi, or Diabroticaundecimpunctata.

Another application wherein the present invention is believed to beparticularly effective is when the pesticide has activity against a weedor a parasitic or saprophytic plant and the transgenic event hasactivity against a member selected from the group consisting ofDiabrotica virgifera, Diabrotica barberi and Diabrotica undecimpunctata.This is believed to be more preferred useful when the weed or aparasitic or saprophytic plant is the African plant known as “Striga”,and even more preferred when the pesticide is ROUNDUP® (available fromMonsanto Company).

In the method of the present invention, thiamethoxam is applied to atransgenic corn seed. Although it is believed that the present methodcan be applied to a transgenic corn seed in any physiological state, itis preferred that the seed be in a sufficiently durable state that itincurs no damage during the treatment process. Typically, the seed wouldbe a seed that had been harvested from the field; removed from theplant; and separated from any cob, stalk, outer husk, and surroundingpulp or other non-seed plant material. The seed would preferably also bebiologically stable to the extent that the treatment would cause nobiological damage to the seed. In one embodiment, for example, thetreatment can be applied to seed corn that has been harvested, cleanedand dried to a moisture content below about 15% by weight. In analternative embodiment, the seed can be one that has been dried and thenprimed with water and/or another material and then re-dried before orduring the treatment with the pesticide. Within the limitations justdescribed, it is believed that the treatment can be applied to the seedat any time between harvest of the seed and sowing of the seed. As usedherein, the term “unsown seed” is meant to include seed at any periodbetween the harvest of the seed and the sowing of the seed in the groundfor the purpose of germination and growth of the plant.

When it is said that unsown seed is “treated” with the pesticide, suchtreatment is not meant to include those practices in which the pesticideis applied to the soil, rather than to the seed. For example, suchtreatments as the application of the pesticide in bands, “T”-bands, orin-furrow, at the same time as the seed is sowed are not considered tobe included in the present invention.

The pesticide, or combination of pesticides, can be applied “neat”, thatis, without any diluting or additional components present. However, thepesticide is typically applied to the seeds in the form of a pesticideformulation. This formulation may contain one or more other desirablecomponents including but not limited to liquid diluents, binders toserve as a matrix for the pesticide, fillers for protecting the seedsduring stress conditions, and plasticizers to improve flexibility,adhesion and/or spreadability of the coating. In addition, for oilypesticide formulations containing little or no filler, it may bedesirable to add to the formulation drying agents such as calciumcarbonate, kaolin or bentonite clay, perlite, diatomaceous earth or anyother adsorbent material. Use of such components in seed treatments isknown in the art. See, e.g., U.S. Pat. No. 5,876,739. The skilledartisan can readily select desirable components to use in the pesticideformulation depending on the seed type to be treated and the particularpesticide that is selected. In addition, readily available commercialformulations of known pesticides may be used, as demonstrated in theexamples below.

The seeds may also be treated with one or more of the followingingredients: other pesticides, including compounds which act only belowthe ground; fungicides, such as captan, thiram, metalaxyl,(methoxam=resolved isomer of metalaxyl), fludioxonil, oxadixyl, andisomers of each of those materials, and the like; herbicides, includingcompounds selected from carbamates, thiocarbamates, acetamides,triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils,phenoxys, ureas, and benzoic acids; herbicidal safeners such asbenzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide,various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone,naphthalic anhydride compounds, and oxime derivatives; fertilizers; andbiocontrol agents such as naturally-occurring or recombinant bacteriaand fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia,Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. Theseingredients may be added as a separate layer on the seed oralternatively may be added as part of the pesticide composition.

Preferably, the amount of the novel composition or other ingredientsused in the seed treatment should not inhibit generation of the seed, orcause phytotoxic damage to the seed.

The pesticide formulation that is used to treat the transgenic corn seedin the present invention can be in the form of a suspension; emulsion;slurry of particles in an aqueous medium (e.g., water); wettable powder;wettable granules (dry flowable); and dry granules. If formulated as asuspension or slurry, the concentration of the active ingredient in theformulation is preferably about 0.5% to about 99% by weight (w/w),preferably 5-40%.

As mentioned above, other conventional inactive or inert ingredients canbe incorporated into the formulation. Such inert ingredients include butare not limited to: conventional sticking agents, dispersing agents suchas methylcellulose (Methocel A15LV or Methocel A15C, for example, serveas combined dispersant/sticking agents for use in seed treatments),polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P),polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVP/VAS-630), thickeners (e.g., clay thickeners such as Van Gel B to improveviscosity and reduce settling of particle suspensions), emulsionstabilizers, surfactants, antifreeze compounds (e.g., urea), dyes,colorants, and the like. Further inert ingredients useful in the presentinvention can be found in McCutcheon's, vol. 1, “Emulsifiers andDetergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.Additional inert ingredients useful in the present invention can befound in McCutcheon's, vol. 2, “Functional Materials,” MC PublishingCompany, Glen Rock, N.J., U.S.A., 1996.

The pesticides and pesticide formulations of the present invention canbe applied to seeds by any standard seed treatment methodology,including but not limited to mixing in a container (e.g., a bottle orbag), mechanical application, tumbling, spraying, and immersion. Anyconventional active or inert material can be used for contacting seedswith pesticides according to the present invention, such as conventionalfilm-coating materials including but not limited to water-based filmcoating materials such as Sepiret (Seppic, Inc., Fairfield, N.J.) andOpacoat (Berwind Pharm. Services, Westpoint, Pa.).

The subject pesticides can be applied to a seed as a component of a seedcoating. Seed coating methods and compositions that are known in the artare useful when they are modified by the addition of one of theembodiments of the combination of pesticides of the present invention.Such coating methods and apparatus for their application are disclosedin, for example, U.S. Pat. Nos. 5,918,413, 5,891,246, 5,554,445,5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coatingcompositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356,5,882,713, 5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103,5,622,003, 5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587,4,383,391, 4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.

Useful seed coatings contain one or more binders and at least one of thesubject combinations of pesticides.

Binders that are useful in the present invention preferably comprise anadhesive polymer that may be natural or synthetic and is withoutphytotoxic effect on the seed to be coated. The binder may be selectedfrom polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinylacetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; fats; oils; proteins, including gelatin and zeins; gumarabics; shellacs; vinylidene chloride and vinylidene chloridecopolymers; calcium lignosulfonates; acrylic copolymers;polyvinylacrylates; polyethylene oxide; acrylamide polymers andcopolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; andpolychloroprene.

It is preferred that the binder be selected so that it can serve as amatrix for the subject pesticides. While the binders disclosed above mayall be useful as a matrix, the specific binder will depend upon theproperties of the combination of pesticides. The term “matrix”, as usedherein, means a continuous solid phase of one or more binder compoundsthroughout which is distributed as a discontinuous phase one or more ofthe subject pesticides. Optionally, a filler and/or other components canalso be present in the matrix. The term matrix is to be understood toinclude what may be viewed as a matrix system, a reservoir system or amicroencapsulated system. In general, a matrix system consists ofpesticides of the present invention and filler uniformly dispersedwithin a polymer, while a reservoir system consists of a separate phasecomprising the subject pesticides, that is physically dispersed within asurrounding, rate-limiting, polymeric phase. Microencapsulation includesthe coating of small particles or droplets of liquid, but also todispersions in a solid matrix.

The amount of binder in the coating can vary, but will be in the rangeof about 0.01 to about 25% of the weight of the seed, more preferablyfrom about 0.05 to about 15%, and even more preferably from about 0.1%to about 10%.

As mentioned above, the matrix can optionally include a filler. Thefiller can be an absorbent or an inert filler, such as are known in theart, and may include woodflours, clays, activated carbon, sugars,diatomaceous earth, cereal flours, fine-grain inorganic solids, calciumcarbonate, and the like. Clays and inorganic solids, which may be used,include calcium bentonite, kaolin, china clay, talc, perlite, mica,vermiculite, silicas, quartz powder, montmorillonite and mixturesthereof. Sugars, which may be useful, include dextrin and maltodextrin.Cereal flours include wheat flour, oat flour and barley flour.

The filler is selected so that it will provide a proper microclimate forthe seed, for example the filler is used to increase the loading rate ofthe active ingredients and to adjust the control-release of the activeingredients. The filler can aid in the production or process of coatingthe seed. The amount of filler can vary, but generally the weight of thefiller components will be in the range of about 0.05 to about 75% of theseed weight, more preferably about 0.1 to about 50%, and even morepreferably about 0.5% to 15%.

The pesticides that are useful in the coating are those pesticides thatare described herein. The amount of pesticide that is used for thetreatment of the seed will vary depending upon the type of seed and thetype of active ingredients, but the treatment will comprise contactingthe seeds with an amount of the pesticide that is pesticidallyeffective. When insects are the target pest, that amount will be anamount of the insecticide that is insecticidally effective. As usedherein, an insecticidally effective amount means that amount ofinsecticide that will kill insect pests in the larvae or pupal state ofgrowth, or will consistently reduce or retard the amount of damageproduced by insect pests.

In general, the amount of thiamethoxam that is applied to the seed inthe treatment will range from about 10 gm to about 2000 gm of the activeingredient of the pesticide per 100 kg of the weight of the seed.Preferably, the amount of pesticide will be within the range of about 50gm to about 1000 gm active per 100 kg of seed, more preferably withinthe range of about 100 gm to about 600 gm active per 100 kg of seed, andeven more preferably within the range of about 200 gm to about 500 gm ofactive per 100 kg of seed weight. Alternatively, it has been found to bepreferred that the amount of the pesticide be over about 60 gm of theactive ingredient of the pesticide per 100 kg of the seed, and morepreferably over about 80 gm per 100 kg of seed.

In preferred embodiments of the present invention the transgenic eventcomprises the ability to express a Cry3Bb.11231 protein or aCry3Bb.11098 protein, and the pesticide is thiamethoxam.

The pesticides that are used in the treatment must not inhibitgermination of the seed and should be efficacious in protecting the seedand/or the plant during that time in the target insect's life cycle inwhich it causes injury to the seed or plant. In general, the coatingwill be efficacious for approximately 0 to 120 days after sowing.

In the subject invention, the pesticides be applied to the seed in theform of a coating. The use of a coating is particularly effective inaccommodating high pesticidal loads, as can be required to treattypically refractory pests, such as corn rootworm, while at the sametime preventing unacceptable phytotoxicity due to the increasedpesticidal load.

Optionally, a plasticizer can be used in the coating formulation.Plasticizers are typically used to make the film that is formed by thecoating layer more flexible, to improve adhesion and spreadability, andto improve the speed of processing. Improved film flexibility isimportant to minimize chipping, breakage or flaking during storage,handling or sowing processes. Many plasticizers may be used, however,useful plasticizers include polyethylene glycol, glycerol,butylbenzylphthalate, glycol benzoates and related compounds. The rangeof plasticizer in the coating layer will be in the range of from bout0.1 to about 20% by weight.

When the pesticide used in the coating is an oily type formulation andlittle or no filler is present, it may be useful to hasten the dryingprocess by drying the formulation. This optional step may beaccomplished by means will known in the art and can include the additionof calcium carbonate, kaolin or bentonite clay, perlite, diatomaceousearth, or any absorbent material that is added preferably concurrentlywith the pesticidal coating layer to absorb the oil or excess moisture.The amount of calcium carbonate or related compounds necessary toeffectively provide a dry coating will be in the range of about 0.5 toabout 10% of the weight of the seed.

The coatings formed with the pesticide are preferably of the type thatare capable of effecting a slow rate of release of the pesticide bydiffusion or movement through the matrix to the surrounding medium.

In addition to the coating layer, the seed may be treated with one ormore of the following ingredients: other pesticides including fungicidesand herbicides; herbicidal safeners; fertilizers and/or biocontrolagents. These ingredients may be added as a separate layer oralternatively may be added in the pesticidal coating layer.

The pesticide formulation may be applied to the seeds using conventionalcoating techniques and machines, such as fluidized bed techniques, theroller mill method, rotostatic seed treaters, and drum coaters. Othermethods, such as spouted beds may also be useful. The seeds may bepresized before coating. After coating, the seeds are typically driedand then transferred to a sizing machine for sizing. Such procedures areknown in the art.

The pesticide-treated seeds may also be enveloped with a filmovercoating to protect the pesticide coating. Such overcoatings areknown in the art and may be applied using conventional fluidized bed anddrum film coating techniques.

In another embodiment of the present invention, a pesticide can beintroduced onto or into a seed by use of solid matrix priming. Forexample, a quantity of the pesticide can be mixed with a solid matrixmaterial and then the seed can be placed into contact with the solidmatrix material for a period to allow the pesticide to be introduced tothe seed. The seed can then optionally be separated from the solidmatrix material and stored or used, or the mixture of solid matrixmaterial plus seed can be stored or planted directly. Solid matrixmaterials which are useful in the present invention includepolyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea,polyacrylate, or any other material capable of absorbing or adsorbingthe pesticide for a time and releasing that pesticide into or onto theseed. It is useful to make sure that the pesticide and the solid matrixmaterial are compatible with each other. For example, the solid matrixmaterial should be chosen so that it can release the pesticide at areasonable rate, for example over a period of minutes, hours, or days.

The present invention further embodies imbibition as another method oftreating seed with the pesticide. For example, plant seed can becombined for a period of time with a solution comprising from about 1%by weight to about 75% by weight of the pesticide in a solvent such aswater. Preferably the concentration of the solution is from about 5% byweight to about 50% by weight, more preferably from about 10% by weightto about 25% by weight. During the period that the seed is combined withthe solution, the seed takes up (imbibes) a portion of the pesticide.Optionally, the mixture of plant seed and solution can be agitated, forexample by shaking, rolling, tumbling, or other means. After imbibition,the seed can be separated from the solution and optionally dried, forexample by patting or air drying.

In yet another embodiment, a powdered pesticide can be mixed directlywith seed. Optionally, a sticking agent can be used to adhere the powderto the seed surface. For example, a quantity of seed can be mixed with asticking agent and optionally agitated to encourage uniform coating ofthe seed with the sticking agent. The seed coated with the stickingagent can then be mixed with the powdered pesticide. The mixture can beagitated, for example by tumbling, to encourage contact of the stickingagent with the powdered pesticide, thereby causing the powderedpesticide to stick to the seed.

The present invention also provides a transgenic corn seed that has beentreated with a pesticide by the method described above.

The treated seeds of the present invention can be used for thepropagation of corn plants in the same manner as conventional treatedcorn seed. The treated seeds can be stored, handled, sowed and tilled inthe same manner as any other pesticide treated seed. Appropriate safetymeasures should be taken to limit contact of the treated seed withhumans, food or feed materials, water and birds and wild or domesticanimals.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

The following examples describe preferred embodiments of the invention.Other embodiments within the scope of the claims herein will be apparentto one skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by the claimswhich follow the examples. In the examples all percentages are given ona weight basis unless otherwise indicated.

EXAMPLE 1

Production of corn seed having a transgenic event effective against cornrootworm and treatment of such seed with thiamethoxam (Cruiser®).

Corn seeds can be prepared to express Bacillus thuringiensis endotoxinCry3Bb.11231 or Cry3Bb.11098 by the methods described for theserespective events in WO 99131248, or in U.S. Pat. No. 6,023,013.

Corn seeds of the same hybrid species, with and without the respectivetransgenic events, can be treated with thiamethoxam (available asCRUISER® from Novartis) as follows:

A seed treatment formulation of the desired pesticide is prepared bymixing a measured amount in water as a carrier and applying theformulation for one minute at room temperature to a measured weight ofcorn seed in a rotostatic seed treater. The respective weights of thepesticide preparation and the corn seed are calculated to provide thedesired rate of treatment of pesticide on the seed. The pesticide ismixed into sufficient water to permit efficient distribution of theformulation to all of the seeds in the batch while minimizing loss oftreatment formulation due to lack of uptake of the formulation by theseeds. Treated seeds are allowed to sit uncapped for at least four hoursbefore planting.

When the seeds are treated with thiamethoxam, a sufficient amount ofCRUISER® Is thoroughly mixed into water to form a seed treatmentformulation, and the formulation Is applied to a weight of corn seed toprovide treatment levels of 300 grams thiamethoxam per 100 kg of seed(.75 mg thiamethoxam/kernel), or 400 grams thiamethoxam per 100 kg ofseed (1.0 mg thiamethoxam/kernel).

EXAMPLE 2

Field trials for the determination of efficacy of transgenic eventCry3Bb.11231 in corn seed in combination with corn root worm pesticideseed treatments against western and northern corn rootworm.

Field trials can be run in accordance with pertinent protocols and inconformance with USDA notification requirements. The purpose of thetrials is to determine the efficacy of transgenic event Cry3Bb.11231 incorn seed in combination with corn root worm seed treatments againstwestern and northern corn root worm.

For each growing site that is selected, the plot design would includedthe following:

Row spacing: 30 inches Plot size: 4 rows × 20 Plant density: 2.0seed/foot Hybrid used: LH198 × LH185 or RX670 Replicates: 4 Design:Randomized complete block Locations: 4 Larvae source: naturalinfestations supplemented by artificial infestation of corn rootwormeggs at 400 eggs/ft (growth stage V2)

The following seed treatment combinations can be used for each growingarea:

Pesticide and amount (grams Al/100 No. Corn Seed Type kg seed or mgai/kernel) 1 Isohybrid None, other than low levels for wire wormprotection 2 Cry3Bb.11231 None, other than low levels for wire wormprotection 3 Cry3Bb.11231 CRUISER ® @ 300 gm Al/100 kg or .75 mgAl/kernel 4 Cry3Bb.11231 CRUISER ® @ 400 gm Al/100 kg or 1.0 mgAl/kernel 5 Isohybrid Force ® 3G @ 0.014 gm Al/m, or 0.15 oz Al/1000 ftrow, applied as a 5″ band on the soil surface at the time of planting. 6Isohybrid Lorsban ® 15G (chlorpyrifos; available from DowElanco) @ 0.11gm Al/m, or 1.2 oz Al/1000 ft row, applied as a 5″ band on the soilsurface at the time of planting.

All seed treatments with pesticides are carried out as described inExample 1. In seed treatment number 1 and 2, Gaucho® (imidacloprid) canbe used for wire worm protection, but at levels sufficiently low that itwould be expected to have no effect on corn rootworms (i.e., at atreatment level of about 60 gm of active/100 kg seed or 0.16 mgactive/kernel), otherwise, seed receiving treatment number 2 should haveonly transgenic event Cry3Bb.11231 and no pesticide treatment that wouldbe expected to be effective against corn rootworm.

For seeds having treatments numbered 3 and 4, the pesticides are appliedby the methods described in Example 1. For seeds having treatmentnumbers 5 and 6, commercially available Force® 3G and Lorsban® 15G areapplied to the soil in a 5″ band at the time of sowing to provide acomparison with standard practices. The levels of application are asshown and are within the ranges recommended for standard commercialpractice.

Corn seeds to be tested should be planted and grown at several differentlocations according to the protocol described above.

The determination of damage by corn rootworm can be made according tothe following protocol. At stage V4-V6, an evaluation of early stand ismade by counting the number of plants per acre. At stage VT-R1, anevaluation of corn rootworm damage can be carried out by methods thatare well known in the industry, and damage by corn rootworm is reportedaccording to the Iowa 1-6 rating system. In that system, the rootsystems of 10 corn plants per plot are recovered and scored using the1-6 rating scale, where: 1=no injury or only a few minor feeding scars,2=feeding injury evident, but no roots eaten back to 1½ inches of theplant, 3=at least one root eaten off to within 1½ inches of the plant,but never an entire node of roots destroyed, 4=one node of roots eatenback to within 1½ inches of the plant, 5=two nodes (circles) of rootseaten back to within 1½ inches of the plant, 6=three nodes (circles) ofroots eaten back to within 1½ inches of the plant.

It is believed that the data that is obtained from these techniqueswould show that transgenic seeds that were treated with thiamethoxamwere more resistant to corn rootworm damage than the transgenic seedswithout such pesticide treatment. Moreover, it is believed that suchtests would show that combination treatments (of transgenic event pluspesticide treatment) were more efficacious that conventional surfacebanding with either FORCE® or LORSBAN®.

The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

1. A method for protecting a transgenic corn plant against feedingdamage by one or more pests, the method comprising providing a seed forthe transgenic corn plant, wherein the seed comprises an exogenous genewhich encodes for the expression of a modified Cry3 protein or Cry3*protein, which differs from wild-type proteins by having specific aminoacid substitutions, additions or deletions as compared with the proteinsproduced by the wild-type organism; and treating the seed with asynergistic amount of thiamethoxam.
 2. The method according to claim 1wherein the Cry3* protein comprises a Cry3B* protein.
 3. The methodaccording to claim 1 wherein the Cry 3B* protein is selected from thegroup consisting of Cry3Bb 11230, Cry3Bb 11231, Cry3Bb 11232, Cry3Bb11233, Cry3Bb 11234 , Cry3Bb 11235, Cry3Bb 11236, Cry3Bb 11237, Cry3Bb11238, Cry3Bb 11239, Cry3Bb 11241, Cry3Bb 11242, and Cry3Bb
 11098. 4.The method according to claim 1 wherein the Cry 3B* protein comprisesCry3Bb11231.
 5. The method according to claim 1 wherein the Cry3B*protein comprises Cry3Bb11098.
 6. The method according to claim 1,wherein the method is capable of reducing the damage caused by cornrootworm so that the damage to transgenic corn is within the range ofabout 15% to about 30% of the damage to non-transgenic corn under thesame conditions, when such damage is expressed as the percent of cornplants having a score of 4-6 as measured by the Iowa Corn Rootworm 1-6Scale.
 7. The method according to claim 6 wherein the method is capableof reducing the damage caused by corn rootworm so that the damage totransgenic corn is within the range of about 20% to about 30% of thedamage to non-transgenic corn under the same conditions, when suchdamage is expressed as the percent of corn plants having a score of 4-6as measured by the Iowa Corn Rootworm 1-6Scale.
 8. The method accordingto claim 1 wherein the seed also has the ability to express at least oneother insecticidal protein that is different from a Cry 3* protein. 9.The method according to claim 1 wherein the seed having the ability toexpress a Cry 3* protein also has a transgenic event that providesherbicide tolerance.
 10. The method according to claim 9 wherein thetransgenic event provides herbicide tolerance against glyphosate. 11.The method according to claim 1 wherein the method has activity againstan insect.
 12. The method according to claim 11 wherein the insect isselected from the group consisting of members of the orders ofLepidoptera, Coleoptera and Hemiptera.
 13. The method according to claim12 wherein the insect is the Coleopteran insect.
 14. The methodaccording to claim 13 wherein the insect is a Diabrotica spp.
 15. Themethod according to claim 14 wherein the insect comprises at least onemember selected from the group consisting of Diabrotica virgifera,Diabrotica barberi and Diabrotica undecimpunctata.
 16. The method ofclaim 1 wherein the synergistic amount of thiamethoxam is from at least100 grams to about 600 grams of thiamethoxam per 100 kilograms of theseed.
 17. The method of claim 16 wherein the effective amount is from atleast 200 grams to about 500 grams of thiamethoxam per 100 kilograms ofthe seed.
 18. The method of claim 1 wherein the amount of thiamethoxamis from at least 70 grams to about 1000 grams of thiamethoxam per 100kilograms of the seed.
 19. The method according to claim 1, wherein themethod is capable of reducing the damage caused by the corn rootworm sothat the damage to the transgenic corn is within the range of about 10%to about 40% of the damage to the non-transgenic corn under the sameconditions, when said damage is expressed as the percent of the cornplants having a score of 4-6 as measured by the Iowa Corn Rootworm 1-6Scale.
 20. A seed of a transgenic corn plant that provides increasedresistance to the resulting corn plant against feeding damage by one ormore pests, wherein the seed comprises an exogenous gene which encodesfor the expression of a modified Cry3 protein or Cry3* protein, whichdiffers from wild-type proteins by having specific amino acidsubstitutions, additions or deletions as compared with the proteinsproduced by the wild-type organism, wherein the seed has been treatedwith a synergistic amount of an insecticide consisting essentially ofthiamethoxam.
 21. The seed according to claim 20 wherein the Cry3*protein is derived from Bacillus thuringiensis.
 22. The seed accordingto claim 21 wherein the Cry3* protein comprises a Cry3Bb* derived fromBacillus thuringiensis.
 23. The seed according to claim 22 wherein theCry3Bb* protein is selected from the group consisting of Cry3Bb 11230,Cry3Bb 11231, Cry3Bb 11232, Cry3Bb 11233, Cry3Bb 11234, Cry3Bb 11235,Cry3Bb 11236, Cry3Bb 11237, Cry3Bb 11238, Cry3Bb. 11239, Cry3Bb 11241,Cry3Bb 11242, and Cry3Bb 11098 protein derived from Bacillusthuringiensis.
 24. A transgenic corn seed that has been treated by themethod comprising: providing a seed for the transgenic corn plant,wherein the seed comprises an exogenous gene which encodes for theexpression of a modified Cry3 protein or Cry3* providing a seed for thetransgenic corn plant, wherein the seed comprises an exogenous genewhich encodes for the expression of a modified Cry3 protein or Cry3*protein, which differs from wild-type proteins by having specific aminoacid substitutions, additions or deletions as compared with the proteinsproduced by the wild-type organism; and treating the seed with asynergistic amount of an insecticide consisting essentially ofthiamethoxam.